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In recent years, Prussian blue nanoparticles (PB-NPs) and theire analogues, that exhibit an increased catalytic activity, have attracted considerable attention in nanotechnology. Such nanoparticles are regarded as an alternative to natural enzymes and are intensively used in biosensorics, diagnostics and experimental biomedicine. Recently, it has been shown that Prussian blue nanoparticles and their analogs, also referred to as “nanozymes”, can be used as active oxygen scavengers and antibacterial or anti-inflammatory drugs due to their polyenzymatic activities, namely oxidase, peroxidase, superoxide dismutase. Their excellent biocompatibility and biodegradability mean that they are ideal for in vivo use. Prussian blue nanoparticles are highly efficient electron transporters that engage in oxidation and reduction activity, which makes them promising mediators and catalysts of reactions. They also show great promise as nanodrug carriers and biological detection sensors due to their large specific surface area, unique chemical characteristics, and variable qualities, which, more importantly, can significantly increase their therapeutic effect. Prussian blue nanoparticles, as therapeutic and diagnostic tools, have achieved significant success in biological nanomedicine. This review is devoted to the methods of synthesis of Prussian blue nanoparticles, the study of their structure, properties and role in the creation of analytical sensors and their promising significance for biomedicine.